The long range goal of this research is to elucidate the catalytic pathway and mechanism of action of the hydroperoxidases horseradish peroxidase, cytochrome c peroxidase and catalase. This includes the identification of the elementary steps in the catalytic pathways of these heme enzymes and the detection and stabilization of the catalytic intermediates produced by each elementary step. These goals are difficult to achieve because many elementary steps are not accessible for study by current techniques under normal conditions. More catalytic intermediates are present at low concentrations for brief periods of time. To overcome these problems, stopped-flow cryoenzymology is being used to detect and stabilize new catalytic intermediates in these reactions, such as the newly discovered horseradish peroxidase compound O. In particular, the interconversion between isoelectronic, high-valent forms of these enzymes during both the initial oxidation of the ferric enzymes with hydrogen peroxide and their subsequent reductions by substrates are under study. The microscopic rate constants for each elementary step will be measured and their temperature dependencies used to characterize the thermodynamic activation parameters for each step and the relative thermodynamic stabilities of each intermediate. Changes in the values of the rate constants for elementary steps with pH will be investigated in order to assess whether they are influenced by the state of protonation of distal and proximal residues with the goal of assigning a catalytic role to these parts of the protein. In certain cases, parallel experiments will be carried out in Nalpha- acetylated microperoxidase-8 and met-myoglobin, since these species can serve as "models" for these hydroperoxidases. Resonance Raman spectroscopy will be used to provide information about structures of the longer lived intermediates. A specific chemical will be formulated for each enzyme that is consistent with the observed catalytic pathway and pathway and the function of the hydroperoxidase.